Efficacy in CB 1 Cannabinoid Receptor Signal Transduction. NIDA November, 2003 Allyn Howlett, Ph.D....

Efficacy in CB1 Cannabinoid Receptor

Signal Transduction

.

NIDANovember, 2003

Allyn Howlett, Ph.D.Neuroscience/Drug Abuse Research ProgramJ. L. Chambers Biomedical/Biotechnology Research InstituteNorth Carolina Central University

Supported by Natl Institute on Drug Abuse

Frontiers in Addiction Research

Cannabinoid Receptor Subtypes

• CB1

– Found in neuronal cells and brain; other non-innervated tissue?

– Regulates neurotransmitter release

• CB1(A)

– Splice variant mRNA found in human brain, but not predicted in rodent gene (Sanofi Recherche)

– Similar pharmacology and signal transduction as CB1

• CB2

– Found in immune tissue (B cells, macrophages, T cells)– Activity not fully characterized

• CB?? or CB?– ? Antinociceptive effects of anandamide in CB1 (-/-) mice (Martin)– ? Vascular effects of anandamide not reproduced by other agonists

(Kunos)

Herkenham et al. (1991) J. Neurosci. 11: 563

CB1 Cannabinoid Receptor

NH2

V

P

A

N

Q

S

T

N

I

Q

F

A

T

S

V

T

V

V

V

V

Y

D

I

A

F

SF

F

D

H

F

S

R

H

V

L

K

F

A

D

N

T

LP

T

V

L

F

F

S

V

S

K

S

V

G

A

N

W

I

M

G

C

L

A

F

L

A

W

K

P

I

C

K

V

T

V

L

V

L

L

I

E

I

L

L

TW

L

MY

F

YA

IY

AA

V

RL

I

K W

A

QT

GK

V

R

T

QV

ST

HSI

I

K DE

H

K

G

V

V

I

T

S

R

P

R H

Q

K A IY

S

D

V

T

S

M

A

L

R

A

L

I

F

Y

R

I

P

H

L

I

A

YI

SF

S

PH

CR

RL

C

H

S

V

R

V

SI

PO4

Q

R

V

S

VL

V

I

C

L

P

DY

FG

P

S

D

S

I

F

T

T

D

M

R

I

MQ

COOH

IL

LD

A

H

PC

LV

AI

R

S

Y

R

IL

WI

VL

K

S

R

L

FD

ML

GI

V

PF

K

A

G

D

VV

A

A

K

S

F

A

T

TP

YR

M

M

ML

G

SN

I

E

N

A

L

S

C

CN

V

T

K

I

FC

L

L

L

IK

VF

L

LLLG

LELLL

L

GL

LG

L

CB1 Cannabinoid Receptor, A G-Protein Coupled Receptor

EC1

EC2EC3

IC1

IC2

IC33D structure recently determined (Biochemistry 2002, 41, 11344)

extracellular

intracellular

Homology Model of the CB1 Receptor

Biopolymers (Peptide Sciences), 2003, 71, 169-189

E2 loopas a part of binding site

extracellular

intracellular

Cannabinoid Receptor Agonists• Classical Cannabinoid

(ABC-tricyclic)

• Nonclassical Cannabinoid (AC-bicyclic; ACD-tricyclic)

– CP55940; CP55244 (Pfizer)

• Aminoalkylindole– WIN55212-2 (Sterling

Research Inst.)

• Eicosanoid– Arachidonylethanolamide

(anandamide)– 2-Arachidonoylglycerol

• Aryl Pyrazole analogs

– Organon analogs (Razdan and Martin)

O H

O H

H OC P 5 5 9 4 0

A

C 123

45

6

7

89

1 0

1 11 2

1 "2 "

3 "

1 '2 '

3 '

4 '

5 '

6 '

7 '8 ' 9 '

O H

O H

H OC P 5 5 2 4 4

A

C 123

45

6

7

89

1 0

1 2

1 51 31 4

1 '2 '

3 '

4 '

5 '

6 '

7 '8 ' 9 '1 7

D

O H

O

T H C

B

C

A

123

45

7

89

1 '2 '

3 '

4 '

5 '

1 0

1 1

W I N 5 5 2 1 2 - 2

NO

O

NO

13

5

8

1 0

A n a n d a m i d e

NH

OO H

2-Arachidonylglyceryl ether (noladin ether)

Some Other EndoCannabinoids

O-Arachidonoyl ethanol ester(Virodhamine)

2-Arachidonoylglycerol(2-AG)

OOH

OH

O2.5 >141

OOH

OH

Devane et al. (1992) Science 258: 1946Mechoulam et al. (1995) Biochem. Pharmacol. 50: 83 Hanus et al. (2001) PNAS 98: 3662Porter et al. (2002) J. Pharmacol. Exp. Ther. 301: 1020

CB1/CB2 Affiniy Ratio

O

OH O

???

CB1 Receptor Signal Via Gi/o Proteins

Signal Transduction Effector G protein Subunit

Ion Channels K+ currents Gi (1,2,3? Via cAMP?)Ca2+ currents Gi or Go beta-gamma?

Mitogen-Activated Protein Kinase Gi (1,2,3?) beta-gamma? or Go(1,2)?

Other?

PLA2 ? Ca2+ mobility? Focal Adhesion Kinase? PI3Kinase? NO synthesis? Sphingomyelin hydrolysis and ceramide?

250

105

50

35

75

N18TG2 Cells

C6 glioma Cells

CB1 R

CP52444

CP55940 9-THC

CBN

CBD

(+)isomers

Adenylyl Cyclase (types 5,6) Gi (1,2,3?)alpha (types 1,3,8 to inhibit? Or types 2,4,7 to stimulate?)

Cannabinoid receptor agonists inhibit N-typeCa2+ currents in differentiated N18 neuroblastoma cells

Mackie et al., Mol.Phm.44:498’93

- + Serum

WIN55212

C6 Glioma Cells

Methanan-damide

CP55940

Cannabinoid Agonist-induced MAPK PhosphorylationSignal Transduction via Gi/o

+ - + - + - Pertussis Toxin

N18TG2 Neuroblastoma Cells

- - - + + + Pertussis Toxin- + Serum

MA WIN CP MA WIN CP

CP55940 and Methanandamide induce Nitric Oxide (NO) production in N18TG2 neuroblastoma cells

Control CP55940

Methanandamide L-NNA + Methanandamide

CB1 Receptor Signaling via G-proteins

• The domains of the CB1 receptor selective for interaction with G-proteins

• Agonists can affect CB1 receptor – G-protein association differentially

• Speculation on conformational induction & G-protein activation

Peptides Synthesized from the IL3 and C-terminal Domains

CB1301 begins IL3;3 peptides span the loop

CB1401 begins at membrane interface, extend beyond cys-palmitoyl anchor

Peptides Derived from the Intracellular CB1 Receptor

GK

ST

HS

I II

K

DEC3

I7.31(375)

F

L

V

L

KI

C

N

F

L

T

CS

K

A

LM

N

F

M

AV

I

V

G

KG

LM

D

WL

IY

CPM

H5.34(270)

T

I

I

G

D

F

F

Y

S

D

V

H

P

WL

C

E

TI

VSQ

L

L

IV

V

P

K

T

L

I

G

AL

VI

NS

YP

T

IV

TL

V

R

I

KV

L

IA

LI

DL

S

VL

V

MY

FL

YA

IL

YA

L

AV

C

RL

I

KW

A

QT

V

RT

QV

P

RA

D

Q

M

COOH

R6.28(336)

LRA

S

K

DAL M

FR FRH

S SP

EC

GT

A

W

H

E

W

K

G

A

C

V

V

N

I

I

T

F

S

M

R

P

R

H

Q

K

V

G

S

A

A

K

I

Y

S

F

S

C2

V

D

R

S

V

L

T

S

M

A

D

F

L

L

R

T

R

N

V

K

A

L

I

T

F

F

A

Y

R

I

P

R

H

L

I

HH

V

F

L

F

S

F

F

A

S

D

Y

D

I

NH2

V

IY

A

S

I

F

V

SV

P

H

S

CR

VA

T

R

Q

T

N

I

F

L

C

QP

C1

S

H

A

S

T

V

R

VN

S

V

I

L

LLLG

LELLL

L

GL

LG

G

GL

E1

E2E3

Extracellular side

Cytoplasmic side

TM1 TM3TM5

TM7N1.28(112)

S1.60(144)

H2.68(181)

R2.37(150)

D3.20(184)

H3.55(219)T4.38(229)

L4.62(253)

I5.61(297)S7.57(401)

Gi1 & Gi2 Gi3 & Go

G6.61(369)

Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts

Peptide 401: - + - + - +

Gi1 Gi2 Gi3

Peptide CB1401 Disrupts the CB1 Receptor Association with Go but not Gi1/2 in Rat Brain Membrane Extracts

Peptide CB1401 Disrupts the Association Between CB1 Receptor and Gi3 but not Gi1 or Gi2 in Rat Brain Membrane Extracts

Peptide 401: - + - + - +

Gi1 Gi2 Gi3

Peptides from IL3 Disrupt the CB1R Association with Gi1 & 2 but not Gi3 in N18TG2 membrane extracts

IL3 peptides: - + - + - +

CB1R

Gi alpha

Conclusions•CB1 Receptor-G alpha complexes exist in the absence of agonists, but can be disrupted by pertussis toxin or GTP analogs.•The juxtamembrane C-terminal domain is involved in the association with Go & Gi3, but not Gi1 & G2.•CB1 IL3 domain is involved in the association

with Gi1 & Gi2 but not Gi3.

Conformational changes in the intracellular surface may direct interaction with selective G proteins

0

20

40

60

80

100

10 100 1000

i1

Log [WIN 55212-2] (nM)

Rel

ativ

e D

ensi

tyG

i/C

B1R

i3 i2

0

20

40

60

80

100

10 100 1000

i2

i1

i3

Log [DALN] (nM)

Rel

ativ

e D

ensi

tyG

i/C

B1R

0

20

40

60

80

100

10 100 1000

i3

i2 i1

Log [Methanandamide] (nM)

Rel

ativ

e D

ensi

tyG

i/C

B1R

Agonist Regulation of Gi/CB1R Association

CP55244 Binding Model Biopolymers (Peptide Sciences), 2003, 71, 169

Assumption: H-bonding between K3.28(192) and phenolic OH

OH

OH

HO +H3NK3.28(192)

blue/green: less lipophilicbrown: more lipophilic

WIN55212-2 Binding Model

aroyl-down1

aroyl-up1

TM2

TM3

TM5

TM7

WIN55212-2 and CP55244 Binding to CB1 Receptor

F5.42(278)V3.32(196)

E(258)

K3.28(192)

CP55244 WIN55212-2

H-bonding:

Y5.39(275)

T5.38(274)

G-protein Activation Mechanism by Receptor Conformational Change

By breaking H-bonding network

By breaking hydrophobic interaction

By breaking H-bonding network

CP55244

WIN55212-2

Response

Phosphorylation by GRK

Arrestin association

Internalization

Conformational Induction of R-G Complex by A

A1

Response 1

A2

Response 2

A3

Response 3

InAInARiGInverse Agonist Response

Agonist Directed “Trafficking” of Signal Transduction

`• Signal transduction pathways will depend

upon the G-proteins and effector pathways present in the cell.

• Domain specificity for G-proteins suggests that induction or selection of different conformations of the CB1 receptor can direct selective signal transduction pathways.

• CB1 receptor signaling through a given pathway may be directed by agonist-specific conformational changes in the receptor.

Prospectus

• Few CB receptor subtypes limits use of pharmacophoric distinctions in ligand affinities to separate therapeutic from untoward effects.

• Can we develop agonists that induce receptor conformations that activate specific G proteins ?

• Manipulation of G protein coupling may promote signal transduction pathways limited to cell types that regulate therapeutic responses.

Collaborators & Acknowledgements

• JLC-BBRI at NCCU

Derek Norford, Skyla Carney, Abdel-Azim Assi

John Joong-Youn Shim

Somnath Mukhopadhyay

CMDNJ-RWJMS William Welsh

J Nehru Univ, Delhi Sudha Cowsik

• $$ National Institute on Drug Abuse

Efficacy in CB 1 Cannabinoid Receptor Signal Transduction. NIDA November, 2003 Allyn Howlett, Ph.D....

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